Purpose:
Several approaches have been developed for gene therapy in RPE65-related Leber congenital amaurosis. To date, all the strategies that have reached the clinical stages rely on adeno-associated viral vectors. We have developed a lentiviral-based strategy of RPE65 gene transfer that efficiently restores protein expression and cone function, as established in mouse models of RPE65 deficiency. The aim of this study was to evaluate the ocular and systemic safety of this lentiviral-based therapy (LV-RPE65) on healthy primates. We report here the retinal imaging results. .

Methods:
Three female Macaca fascicularis received one subretinal injection containing LV-RPE65 (2 animals) or the vehicle (1 animal), using a 41 Gauge cannula. The contralateral eyes of treated animals served as additional controls by receiving either the vehicle (1 animal) or no treatment (1 animal). The bleb (100µl) extended to the posterior pole detaching the macula. Two additional female monkeys will receive a higher dose of LV-RPE65 in one eye and the vehicle in the contralateral control eye. Funduscopy, OCT, fundus autofluorescence, Fluorescein and ICG angiography, anterior chamber cell count, and electroretinography were performed at baseline and at different time points. Blood, urine and tears were regularly sampled to evaluate LV-RPE65 biodistribution and the immune response. Histological analysis and PCR will be performed on specific organs.

Results:
In eyes injected with the vehicle, OCT showed that the subretinal bleb was reattached within 7 days. In eyes injected with LV-RPE65 the transient detachment was associated with an initial shortening of the photoreceptor outer segments. An area of depigmentation was observed on funduscopy, corresponding to the initial bleb. A particular pattern around the retinal vasculature, suggestive of gyrate angeitis was observed shortly after injection in all eyes that had received the vector but not in the control eye. This modification resolved with time; further analysis will help to determine the nature of this reaction.

Conclusions:
Subretinal injection of LV-RPE65 induces an early retinal reaction in Macaca fascicularis. Although transient, this early response has to be better characterized in order to safely translate this approach to RPE65-deficient human retinas.